Various biosynthetic products are produced in cells via natural metabolic processes and are used in many branches of industry, including the foodstuffs, feedstuffs, cosmetics, feed, food and pharmaceutical industries. These substances, which are summarily referred to as fine chemicals/proteins, comprise, inter alia, organic acids, both proteinogenic and nonproteinogenic amino acids, nucleotides and nucleosides, lipids and fatty acids, diols, carbohydrates, aromatic compounds, vitamins and cofactors, and also proteins and enzymes. They are most conveniently produced on a large scale by growing bacteria strains or other microorganisms which have been developed in order to produce and secrete large quantities of the substance desired in each case. Organisms particularly suitable for this purpose are coryneform bacteria, Gram-positive nonpathogenic bacteria.
It is known that amino acids can be prepared by fermentation of strains of coryneform bacteria, in particular Corynebacterium glutamicum. Due to the great importance, continuous work is carried out on improving the production processes. Process improvements may relate to fermentation technique measures such as, for example, stirring and oxygen supply, or to the composition of the nutrient media, such as, for example, the sugar concentration during fermentation, or to the work-up to obtain the product, for example by ion exchange chromatography or else spray drying, or to the intrinsic performance properties of the microorganism itself.
Methods of recombinant DNA technology have likewise been employed for some years to improve Corynebacterium strains producing fine chemicals/proteins, by amplifying individual genes and studying the effect on the production of fine chemicals/proteins.
Other ways of developing a process for producing fine chemicals or proteins or of increasing or improving the productivity of an already existing process for preparing fine chemicals or proteins comprise increasing or altering expression of one or more genes and/or influencing translation of an mRNA by way of suitable polynucleotide sequences. In this context, influencing may comprise increasing, reducing or else other parameters of the expression of genes, such as time-related expression patterns.
Various components of bacterial regulatory sequences are known to the skilled worker. A distinction is made between the binding sites of regulators, also known as operators, the binding sites of RNA polymerase holoenzymes, also known as −35 and −10 regions, and the binding site of ribosomal 16S-RNA, also known as ribosomal binding site (RBS) or else Shine-Dalgarno sequence.
The composition of the polynucleotide sequence of the Shine-Dalgarno sequence and the sequence of the bases, but also the distance of a polynucleotide sequence present in the Shine-Dalgarno sequence to the start codon are described in the literature (E. coli and S. typhimurium, Neidhardt F. C. 1995 ASM Press) as having a substantial influence on the rate of translation initiation.
Nucleic acid sequences having promoter activity can influence the formation of mRNA in different ways. Promoters whose activities are independent of the physiological growth phase of the organism are referred to as constitutive. Other promoters in turn respond to external chemical as well as physical stimuli, such as oxygen, metabolites, heat, pH, etc. Others in turn display a strong dependence of their activity in different growth phases. Examples of promoters which exhibit a particularly pronounced activity during the exponential growth phase of microorganisms, or else exactly in the stationary phase of microbial growth, are described in the literature. Both characteristics of promoters may have a beneficial influence on the productivity for production of fine chemicals and proteins, depending on the metabolic pathway.
Those nucleotide sequences which may be utilized for increasing or attenuating gene expression have already been isolated in Corynebacterium species. These regulated promoters may increase or reduce the rate at which a gene is transcribed, as a function of the internal and/or external conditions of the cell. In some cases, the presence of a particular factor, known as inducer, may stimulate the rate of transcription from the promoter. Inducers may influence transcription from the promoter directly or else indirectly. Another class of factors, known as suppressors, is capable of reducing or else inhibiting transcription from the promoter. Like inducers, suppressors may also act directly or indirectly. However, thermally regulated promoters are also known. Thus, a rise of the growth temperature above the normal growth temperature of the cell may increase or else attenuate the level of transcription of such promoters.
A small number of promoters from C. glutamicum have been described to date. The promoter of the C. glutamicum malate synthase gene was described in DE-A-44 40 118. This promoter was placed upstream of a structural gene coding for a protein. After transformation of such a construct into a coryneform bacterium, the expression of the structural gene downstream of the promoter is regulated. The expression of the structural gene is induced as soon as a corresponding inducer is added to the medium.
Reinscheid et al., Microbiology 145:503 (1999), have described a transcriptional fusion between the pta-ack promoter from C. glutamicum and a reporter gene (chloramphenicol acetyltransferase). C. glutamicum cells containing such a transcriptional fusion exhibited increased expression of the reporter gene when grown on acetate-containing medium. By comparison, transformed cells growing on glucose showed no increased expression of said reporter gene.
Patek et al., Microbiology 142:1297 (1996), described some C. glutamicum DNA sequences which are able to enhance expression of a reporter gene in C. glutamicum cells. These sequences were compared to one another in order to define consensus sequences for C. glutamicum promoters
Further C. glutamicum DNA sequences which may be utilized for regulating gene expression have been described in WO 02/40679. These isolated polynucleotides are expression units from Corynebacterium glutamicum, which may be utilized either for increasing or else for reducing gene expression. This printed publication furthermore describes recombinant plasmids on which said expression units from Corynebacterium glutamicum are associated with heterologous genes. The method described herein, of fusing a Corynebacterium glutamicum promoter to a heterologous gene, may be employed, inter alia, for regulating the genes of amino acid biosynthesis.
The older, not previously published patent applications DE-A-103 59 594, DE-A-103 59 595, DE-A-103 59 660 and DE-A-10 2004 035 065 disclosed special single promoters from C. glutamicum. 